Fleas are insects which behave as ectoparasites for birds and mammals. They are a serious nuisance both in the raising of animals which are sources of food and fiber and in the nurture of pet cats and dogs. The problem in the latter situation is particularly serious because the infestation also becomes a source of annoyance for the pet owner who may find his or her home generally contaminated with fleas which feed on the pets, and these parasites can induce an allergic reaction in both the pets and humans. The prevalence of flea allergic dermatitis (FAD) constitutes the foremost veterinary dermatological problem in the U.S. (Kwochka, K. W., Vet Clin. North Am (1987) 7:1235-1262). Furthermore, the life cycle of the flea favors a survival of intermediate stages since the adult flea feeds and copulates frequently and contaminates the entire environment with eggs (Nesbitt, G. H., et al., J Am Vet Med Assoc (1978) 173:282-288; Soulsby, E. J. L., in Helminths, Arthropods and Protozoa of Domesticated Animals, 7th ed. (1982), Lea and Febiger, eds., Philadelphia, Pa., pages 378-384).
Although flea (i.e., insect) parasitism can be distinguished from that associated with other parasites, such as helminths, which are worms, and ticks, which are arachnids, control of all forms of parasitism has generally involved internal or external applications of chemicals. Commonly encountered approaches to control of the flea problem are generally focused on use of insecticides in formulations such as sprays, shampoos, dusts, dips, or foes, or in pet collars. None are notably successful. While some of these products are efficacious, they are often not successful in reducing flea populations on the pet or in the home for one or more of the following reasons: (1) failure of owner compliance (frequent administration is required), (2) behavioral or physiological intolerance of the pet to the pesticide product or means of administration, and (3) the emergence of flea populations resistant to the prescribed dose of pesticide. In addition, some flea-control clients are adverse to the use of certain chemicals in their home or on their pet that will remain as residual contaminants in the environment.
Efforts to find nontoxic approaches to flea control have resulted in the recent introduction of insect growth regulators (IGRs) such as methoprene which mimic flea hormones and affect flea larval development. A vaccine to reduce flea infestation on the pet and in the home would constitute another nonchemical approach to flea control and would avoid many of the compliance issues necessary for correct pesticide or IGR administration.
A number of attempts have been made to provide a vaccination approach to endoparasites, to other ectoparasites, or to ectoparasites in general. Danish patent 2644149 (1978) suggests the use of antigenic extracts from intermediate hosts in order to prepare antiparasitic vaccines. The focus of this work is with respect to Schistosomes. PCT application WO88/01277 to Australian National University discloses recombinant DNA encoding a helminth parasite antigen and suggests its use in constructing vaccines. PCT application WO 87/05513 (U.S. Pat. No. 4,814,170) assigned to Aphton Corporation describes antiendo- or antiectoparasite vaccines in general, which are derived from endocrine products, such as juvenile hormones.
PCT application WO 86/02839 suggests vaccines against helminths which contain suspensions, homogenates or extracts of nonparasitic nematode species. Presumably, these nonparasitic species are closely enough related to the parasitic forms to engender appropriate antibodies. A similar approach with respect to protozoa is disclosed in PCT application WO 83/03199. British application 1580539A published in 1980 suggests an antiparasitic vaccine derived from secretions of the parasite. None of these approaches are specifically directed to protection against flea infestation.
The problem of flea allergic dermatitis (FAD) has, however, inspired considerable study of the immunological response of hosts to flea antigens which are, presumably, made available for host exposure through the saliva. These studies focus on the nature of the immune response in the host to these antigens, which do not necessarily result in protection of the host against infestation (Halliwell, R. E. W., J Immunol (1973) 110:442-430; Halliwell, R. E. W., et al., J Allerg Clin Immunol (1978) 62:236-242; Halliwell, R. E. W., et al., Vet Immunol Immunopathol (1985) 8:215-223; Wikel, S. K., Vet Parasitol (1984) 14:321-329). An early study which used FAD as a criterion for response to flea antigens, conducted in 1939, suggested that the FAD response, however, might somehow protect the host in such a way so as to prevent flea bites (Cherney, L. S., et al., Am J Trop Med (1939) 19:327-332).
Others have suggested this general concept with regard to other parasites such as ticks and mosquitoes where it is considered that the hypersensitivity generated by salivary antigens may provide some protection against feeding of the parasite (Ribelto, J. M. C., Ann Rev Entomol (1987) 32:463-478; Brown, S. J., Vet Parasitol (1988) 29:235-264; Wikel, S. K., Vet Parasitol (1988): 29:235-264). Since one of the features needed in an effective antiflea vaccine, especially in the context of protecting pets against flea infestation, is the incentive for owner compliance, it is clearly undesirable for any vaccine to behave in such a way as to correlate efficacy with hypersensitivity and dermatitis.
An alternate concept, that of using "hidden" antigens, has been extensively studied for defense against ticks, e.g., Boophilus microplus (Opdebeeck, J. P., et al., Immunol (1988) 63:363-367; Opdebeeck, J. P., et al., Parasite Immunol (1988) 10:405-410; Wong, J. Y. M., and Opdebeeck, J. P., Immunol (1989) 66:149; Opdebeeck, J. P., et al., Immunol (1989) 67.:388; European patent application 208,507, published Jan. 14, 1987. See also Willadsen, P., et al., J Immunol (1989) 143:1346-1351; Rand, K. N., et al., Proc Natl Acad Sci (U.S.A.) (1989) 86:9657-9661; PCT application WO 88/3929, published Jun. 2, 1988; Kemp, D. H., et al., Int J Parasitol (1986) 16:115-120). This approach is also reviewed by Wikel, S. K., (Vet Parasitol (1988) (supra)). This approach has also been attempted using the thoracic muscles of the stable fly (Schlein, Y., et al., Physiolog Entomol (1976) 1:55-59). Furthermore, it has been shown that IgG components from the host can be found intact in the body cavity of the parasitic insect which indicates the passage of immunogobulin through the midgut wall (Chinzei, Y., et al., Med Vet Entomol (1987) :409-416; Hatfield, P. R., Med Vet Entomol (1988) 2:339-345; Vaughan, J. A., et al., J Med Entomol (1988) 25:472-474). While this phenomenon of immunogobulin passage through the gut wall has also been shown in fleas, the work related not to antibodies to flea antigens, but rather to antibodies formed to rickettsia organisms carried in the flea (Azad, A. F., et al., Am J Trop Med Hyg (1987) 37:629-635).
More recently, a thesis prepared in support of the award of a Ph.D. degree at the University of London by Hatfield, P. R. (1986), investigated the immunization of hosts with homogenates or crude extracts of mosquitoes and fleas. The study showed that fleas that fed on mice immunized with flea homogenates and that ingested flea-specific antibodies showed a significant increase in mortality. Similar results were obtained for mosquitoes. The flea aspect of the study used X. cheopis and utilized a solubilized extract of whole body homogenates prepared from newly hatched, unfed fleas.
Copending application U.S. Ser. No. 07/571,257, filed Aug. 22, 1990, now abandoned, describes the preparation of antiflea vaccines from the membranous elements of fed and unfed flea populations. Both full body membrane fractions and midgut membrane fractions were employed.
The present invention, in contrast, utilizes supernatant flea antigens, preferably from the fed midgut. The antigens in the supernatant provide nonallergic vaccines that are capable of immunizing conventional hosts against flea infestation, and furthermore raise antibodies which can be used for passive immunization.